problems in lentiviral transduction - time to collect, ratio for transfection (Apr/13/2011 )

hi guys, i am new to this experiment so i met lots of problems and want you to help me.
1. i insert my target gene, a 2.5kb fragment into pCSII-EF-IRES-hrGFP, which is about 10.4 kb. finally i got positive clones and confirmed them by enzyme digest. however, when i tried to get midi-preps i failed, nearly no DNA extracted. someone suggested me re-transform ecoli and pick the colony and directly do midiculture. i dont know why.
2. at what time point to collect supernatant containing viral particles? 36h? 48h? 60h? 72h? once or twice? if i save the supernatant at 4c for one night, is it ok for the activity of virus? what is the peak for virus production? is it true that virus will be inactivated at 37c for 24hrs?
3. I used pCMV-delta8/9 as packaging plasmid and pVSV-G as envelope plasmid, and the ratio is 10:9:1 (pCSII:packaging:envelope). I dont know if it is correct. I saw other ratios and dont understand what is the difference.
a lot of questions, sorry. thanks in advance.

-gyma-

gyma on Wed Apr 13 07:08:43 2011 said:

hi guys, i am new to this experiment so i met lots of problems and want you to help me.
1. i insert my target gene, a 2.5kb fragment into pCSII-EF-IRES-hrGFP, which is about 10.4 kb. finally i got positive clones and confirmed them by enzyme digest. however, when i tried to get midi-preps i failed, nearly no DNA extracted. someone suggested me re-transform ecoli and pick the colony and directly do midiculture. i dont know why.CSII plasmid contains LTR which is easy to cause recombinations in Ecoli and makes it hard to get plasmid in high-copy competent cells, low-copy competent cells is preferred. However, I used high-copy DH5a by direct midiculture and successfully got the plasmid. But I still dont know why CSII vector with insert is much harder to amplify than vector only.
2. at what time point to collect supernatant containing viral particles? 36h? 48h? 60h? 72h? once or twice? if i save the supernatant at 4c for one night, is it ok for the activity of virus? what is the peak for virus production? is it true that virus will be inactivated at 37c for 24hrs?first time, I collected at 48h and 72h and 48h is better than 72h. Because my 293FT cells started to die in 24h, so this time I decided to collect at 24h and 48h.
3. I used pCMV-delta8/9 as packaging plasmid and pVSV-G as envelope plasmid, and the ratio is 10:9:1 (pCSII:packaging:envelope). I dont know if it is correct. I saw other ratios and dont understand what is the difference.I found a 4:3:1 ratio seems to be mostly used so I used this. I think this is to keep the molar ration to 1:1:1, but I am not sure.

I got some progress on those questions but I might not be correct. If you know the answer, please do tell me. thanks.